Actin is the most ubiquitous and conserved eukaryotic protein, and plays a central role in processes from muscle contraction to metastasis to bacterial pathogenesis. As a result, a very large number of other proteins interact with actin. These interactions are important in the nucleation and depolymerization of F-actin, in the bundling of F-actin into higher order structures, and in both muscle and non-muscle motility. We have been actively exploring the multiplicity of structural states within F-actin, as well as investigating the polymorphisms in the binding of many proteins to F-actin. These studies have provided new clues about the basis for extremely conserved actin sequences over large evolutionary distances. We propose to greatly extend our current studies by generating three-dimensional reconstructions of F-actin and complexes between F-actin and other proteins at a significantly higher resolution. This has only become possible recently with new computational tools for the reconstruction of polymorphic and weakly scattering filaments imaged by cryo-EM in ice. These higher resolution reconstructions will illuminate the conformational changes between existing G-actin crystal structures and the protomer within F-actin, as well as give information about how subunit-subunit contacts change as subunits twist and tilt in the filament. We will test the hypothesis that the multiplicity of interactions observed for proteins such as villin, dematin, fimbrin, a-actinin and calponin with F-actin is governed by how the actin filament is nucleated. The rationale for this is that within a cell no actin filaments are self-nucleated, and we and others have shown that nucleating actin with another protein can change the structure of an entire actin filament. We will also gain insights into acto-myosin motility by studying a chimeric actin assembled from both yeast and muscle sequences. The regulation of striated muscle contraction involves the troponin complex on the thin filament, and we will help constrain models of the thin filament by studying a complex of actin with Tnl. Overall, these studies will have a significant impact in areas as diverse as muscle contraction, infectious diseases, and cancer biology.